Project Summary: Substantial brain blood flow reductions are observed in Alzheimer?s disease (AD) patients and likely contribute to the cognitive symptoms of the disease. Recent work using in vivo multiphoton imaging has shown that neutrophil adhesion in brain capillaries is a cellular mechanism that causes reduced brain blood flow in AD mouse models, and has further shown that cognitive function is rapidly improved when neutrophil adhesion is blocked to increase brain blood flow. These results suggest that improved understanding of the nature and molecular causes of the vascular inflammation in the AD brain could suggest novel therapeutic strategies, complementary to anti-Amyloid and other treatment approaches currently in development, that aim to improve brain blood flow and potentially reduce cognitive symptoms of AD. In this request for supplemental funding, the aim is to pursue two important extensions of the existing grant. The first goal is to assess the impact of the clean environment the mouse models live in. The effect that underlies the decreased brain blood flow in AD mice is subtle ? about 2% of brain capillaries are not flowing due to an adhered neutrophil. Several studies have shown that immune and inflammatory cell behavior in laboratory mice is altered when those mice are exposed to the flora of wild mice. AD and wild-type mice will be exposed to the flora of mice from pet shops, with controls kept in standard laboratory housing conditions. The incidence and cause of capillary stalling and the impact of capillary stalls on brain blood flow will be compared between these groups to determine if altered inflammatory cell phenotypes due to the clean environment of laboratory mice influences the capillary stalling phenomena that underlies brain blood flow deficits in AD mice. This work extends Aim 1 of the original proposal, which seeks to characterize the incidence and cause of non-flowing capillaries in AD mice. The second goal relates to increasing our understanding of the vascular inflammation that leads to increased neutrophil adhesion in capillaries in the AD mice. Recent work has shown that inhibition of NOX2-containing NAPDH oxidase, a reactive oxygen producing enzyme, decreases the incidence of capillary stalling in AD mice. Here, the proposal is to extract brain endothelial cells from AD and wild-type mice with and without inhibition of NAPDH oxidase, and use RNA sequencing to determine differences in gene expression between these four groups. Newly developed approaches that do not overly activate the endothelial cells during extraction and processing will be utilized. Such work to clarify the details of the inflammatory response is critically important for identifying a potential drug target to block this effect and improve brain blood flow in patients. Long term, systemic inhibition of neutrophil adhesion could have hard to manage side effects, and this gene expression data will help to identify a place to intervene that is more brain endothelia and AD specific. This work extends Aim 2 of the original proposal, which aims to identify the molecular signaling that leads to increased capillary stalling in AD mice.